Aiguo Zhao, Ruiwen Ding, Cheng Wang, Cheng Chen, Dongmei Wang
College of Forestry, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi 712100, China.
Key Laboratory of Exploitation and Utilization of Economic Plant Resources in Shaanxi Province, No. 3 Taicheng Road, Yangling, Shaanxi 712100, China.
Tree Physiol. 2023 Jan 5;43(1):169-184. doi: 10.1093/treephys/tpac101.
Accumulation of anthocyanins largely determines the fruit color, and dihydroflavonol 4-reductase (DFR) is a key enzyme involved in the formation of anthocyanins. However, the catalytic and regulatory mechanisms of DFR are unclear. In this study, the gene encoding DFR from Zanthoxylum bungeanum Maxim. was cloned and ZbDFR was analyzed in detail. The ZbDFR accepted dihydrokaempferol, dihydroquercetin and dihydromyricetin as substrates. Flavonols such as myricetin, quercetin and kaempferol significantly inhibited the activity of ZbDFR, while quercitrin and isoquercitrin slightly increased the activity. Quercetin was a competitive inhibitor at low concentrations, and it had a combined effect of competitive and noncompetitive inhibition at high concentrations, which was consistent with ZbDFR having two inhibitor binding sites. In addition, the content of different types of flavonoids in Z. bungeanum peel at green, semi-red and red stage was analyzed, and the in vivo results could be explained by the regulation of ZbDFR activity in vitro. Site-directed mutagenesis combined with enzyme activity experiments showed that Ser128, Tyr163, Phe164 and Lys167 are the key catalytic amino acid residues. The Ser128, Tyr163 and Lys167 were crucial for the hydrogen transfer reaction, and mutation of these amino acids resulted in the loss of all or most of the activity. Phe164 was found to be important for the regulation of ZbDFR by flavonols. Accordingly, ZbDFR is a node at which flavonoids regulate the synthesis of anthocyanins and proanthocyanins.
花青素的积累在很大程度上决定了果实颜色，而二氢黄酮醇4-还原酶（DFR）是参与花青素形成的关键酶。然而，DFR的催化和调控机制尚不清楚。在本研究中，克隆了花椒（Zanthoxylum bungeanum Maxim.）中编码DFR的基因，并对ZbDFR进行了详细分析。ZbDFR接受二氢山奈酚、二氢槲皮素和二氢杨梅素作为底物。杨梅素、槲皮素和山奈酚等黄酮醇显著抑制ZbDFR的活性，而槲皮苷和异槲皮苷则略微增加其活性。槲皮素在低浓度时为竞争性抑制剂，在高浓度时具有竞争性和非竞争性抑制的联合作用，这与ZbDFR具有两个抑制剂结合位点一致。此外，分析了花椒果皮在绿色、半红色和红色阶段不同类型黄酮类化合物的含量，体内结果可以通过体外ZbDFR活性的调控来解释。定点诱变结合酶活性实验表明，Ser128、Tyr163、Phe164和Lys167是关键的催化氨基酸残基。Ser128、Tyr163和Lys167对氢转移反应至关重要，这些氨基酸的突变导致全部或大部分活性丧失。发现Phe164对黄酮醇对ZbDFR的调控很重要。因此，ZbDFR是黄酮类化合物调节花青素和原花青素合成的一个节点。
